Cells use their surface receptors to monitor and respond to subtle changes in the composition of their immediate environment. Drugs which block or activate specific receptors can therefore be used to modify cellular functions for medicinal purposes.
During the last two or three decades some of the most profitable drug discoveries have resulted from the detailed analysis of ligand-receptor interactions and the development of simple assays which have been used to screen for compounds that block ligand binding or directly stimulate receptors. Molecular biology has now uncovered a plethora of high molecular weight polypeptide ligands (for examples, see WO 94/27643 [Targeted Genetics Corporation] and references therein) with diverse biological activities and the Human Genome Project promises to uncover many more. Thus, a major challenge for the pharmaceutical industry is to discover new drugs that block or mimic the effects of these ligands, or exhibit greater specificity for receptor subtypes (Luyten & Leysen 1993 Trends in Biotechnology 11, p247).
The process of drug discovery is relatively simple in principle (Hodgson 1992 Bio/Technology 10, p973). Large numbers of compounds are screened using assays that can detect the desired biological activity and "lead" compounds identified in this way are then optimised to meet development criteria (Bevan et al., 1995 Trends in Biotechnology 13, p115; Ecker & Crooke 1995 Bio/Technology 13, p351).
The likelihood of identifying (and successfully optimising) a lead compound would be increased by the use of simple, sensitive assays that can tolerate a high throughput of drug candidates per unit time and by screening as many different compounds as possible.
There are many types of binding assay available, each with its own advantages and disadvantages. Non-equilibrium assays are suitable for detection of high affinity binding reactions whereas equilibrium assays, such as the scintillation proximity assay (Bosworth & Towers, 1989 Nature 341, p167) are required for detection of low affinity binding reactions characterised by rapid dissociation of bound ligand. Assays such as these, that require a pure source of receptor protein, are problematic because it remains difficult to purify membrane receptor proteins in large quantity (Schertler 1992, Curr. Opin. Struct. Biol. 2, p534). Cell-based assays are therefore preferred because most receptors can be expressed easily from cloned DNA.
Usually the ligand is obtained in pure form and labelled, such that it can be easily detected after it has bound to its cognate receptor on the surface of a mammalian cell. Binding to the receptor is monitored by detection of label on the target cells and lead compounds are identified by virtue of their ability to reduce the amount of receptor-bound label. However, it can be problematic to obtain a pure source of the ligand, and attaching a label (e.g. radioiodine, fluorescein, biotin) to the ligand can alter its affinity for the receptor.
Alternatively, the ligand may be attached to a toxic moiety such that binding to the receptor causes the target cells to die; the lead compound is then detected by its ability to prevent the death of the target cells. However, this type of assay is unlikely to detect a typical lead compound with weak blocking activity, insufficient to prevent the delivery of at least some toxin to the target cells.
In other cell-based assays the binding of a ligand to its receptor is detected by measuring a biochemical or physiological response of the target cell. One of the pitfalls of this type of assay is that a compound may block the response of the target cell in other ways than by interfering with ligand binding, giving rise to a large number of false positives.
Ligand-dependent, Receptor-mediated Retroviral Sequestration
Retroviral envelope glycoproteins mediate specific viral attachment to cell surface receptors and subsequently trigger fusion between the viral envelope and the target cell membrane. Retroviral envelope glycoproteins consist of an external glycoprotein moiety (SU) noncovalently attached at its C-terminus to a smaller transmembrane polypeptide moiety (TM). Each surface projection (or spike), visible by electron microscopy on the viral surface, is a trimer of identical envelope glycoprotein subunits. SU comprises two domains connected by a proline-rich hinge, the N-terminal domain conferring receptor specificity and exhibiting a high degree of conservation between murine leukaemia viruses (MLVs) with different host ranges (Battini et al., 1992 J. Virol. 66, p1468-1475).
A general method has been disclosed that allows the display of a polypeptide ligand (which may be glycosylated) on the surface of a retroviral vector as a genetically encoded extension of the viral envelope protein (WO94/06920, Medical Research Council). The engineered retroviral vector then adopts the binding specificity of the displayed ligand. To date several different polypeptide ligands have been displayed on murine leukaemia virus (MLV)-based retroviral vectors, including single chain antibodies, cellular growth factors and immunoglobulin binding domains (WO94/06920 Medical Research Council; Cosset et al., 1994 Gene Therapy 1 pS1; Nilson et al., 1994 Gene Therapy 1, pS17). In principle, this technology should allow the display of many different structural classes of binding domains on retroviral vectors, including glycopolypeptides and glycoproteins.
The present inventors, in collaboration with colleagues, have also recently discovered a novel biological phenomenon called ligand-dependent, receptor-mediated viral sequestration (illustrated in example 1). A polypeptide ligand is fused (by genetic engineering) to the envelope protein of an MLV-based retroviral vector such that the envelope protein to which it has been grafted remains substantially intact and capable of binding to its natural receptor, and the fused non-viral polypeptide ligand is displayed on the viral surface. The virus displaying the fused non-viral polypeptide ligand is then capable of multivalent attachment to the natural virus receptor and to the cognate receptor for the non-viral ligand; attachment to the natural virus receptor leads to infection of the target cell, whereas attachment to the cellular receptor for the displayed non-viral ligand may not lead to infection of the target cell. Where the target cell expresses both species of receptor and attachment through the displayed non-viral ligand does not lead to infection, the two binding reactions (envelope protein to natural receptor and non-viral ligand to its cognate receptor) proceed in competition and the infectivity of the virus for the target cells is reduced in proportion to the efficiency with which the second binding reaction competes virus away from the natural virus receptor.
For example, when epidermal growth factor (EGF) was displayed on an amphotropic retroviral vector, the engineered vector bound preferentially to EGF receptors present on EGF receptor-positive human cells and gene transfer did not occur. EGF receptor-negative cells were fully susceptible to the engineered retroviral vector but showed reduced susceptibility when they were genetically modified to express EGF receptors. The reduction in susceptibility was in proportion to the level of EGF receptor expression. Moreover, when soluble EGF was added to competitively inhibit virus capture by the EGF receptors, gene transfer was restored. The loss of infectivity of the EGF-displaying virus on EGF receptor expressing cells is believed to reflect a block to membrane fusion between the viral envelope and the target cell plasma membrane.
The degree to which gene transfer is inhibited depends, at least in part, on the relative affinities of the two binding reactions (envelope protein to natural receptor and non-viral ligand to its cognate receptor), the relative densities of the two receptors on the target cell surface, and the relative densities of the non-viral ligand and the intact envelope protein on the viral surface. Inhibition of gene transfer is additionally influenced by intrinsic properties of the receptor for the non-viral ligand, such as the distance it projects from the target cell membrane, its mobility within the target cell membrane and its half life on the cell surface after engagement of ligand.
The present invention makes use of these discoveries to provide the basis for a new assay method, ideally suited to the screening of compounds which may affect the binding between a ligand and its receptor.